Liposomes containing oligonucleotides

ABSTRACT

It is possible to radiosensitize tumor cells by administration of compositions containing the Human antisense c-raf- 1  oligodeoxyribonucleotide (ODN/oligo) sequence:  5 ′-GTGCTCCATTGATGC- 3 ′ (seq. # 1 ) wherein only the end bases are phosphorylated is a preferred embodiment. Antisense sequences of up to 40 bases which containing this sequence may be used in accord with the teachings of this disclosure. Compositions comprising a cationic liposome of dimethyldioctadecyl ammonium bromide, phosphatidylcholine and cholesterol may be used as a carrier system. The liposomes provide a new carrier system that is particularly useful for administration of sequences for therapy.

[0001] This application takes priority from Provisional application Ser.No. 60/041,192 filed Mar. 21, 1997. This work was supported by grantsfrom the National Institutes of Health. The United States Government hascertain rights in this invention.

FIELD OF THE INVENTION

[0002] This invention is related to use antisense of sequences of ≦40bases for enhancing radiosensitivity of radiation-resistant tumors andto cationic liposomes which are particularly useful as carriers forantisense sequences.

BACKGROUND OF THE INVENTION

[0003] Radiation therapy is an important treatment modality of cancer.However, therapeutic management may be limited by the inherent relativeresistance of some cancers to the cytotoxic effects of ionizingradiation. Recently, several lines of investigation have coalesced todemonstrate a link between certain oncogenes (ras, raf, cot, mos, myc),growth factors (PDGF, FGF) and the phenomenon of cellular resistance toionizing radiation.

[0004] It was previously reported that expression of antisense c-raf-1cDNA results in reduced expression (RNA) of c-raf-1 gene, a cause ofdelayed tumor growth in athymic mice and in enhanced radiationsensitivity of relatively radioresistant laryngeal squamous carcinomacells, SQ-20B (Kasid et al., Science 243:1354-1356, 1989).

SUMMARY OF THE INVENTION

[0005] It is possible to radiosensitize tumor cells by administration ofcompositions containing the Human antisense c-raf-1oligodeoxyribonucleotide (ODN/oligo) sequence: 5′-GTGCTCCATTGATGC-3′(seq. #1) wherein only the end bases are phosphorylated is a preferredsequence. Antisense sequences of up to 40 bases which containing thissequence may be used in accord with the teachings of this disclosure. Acomposition of the 25-mer oligo:

[0006] 5′-CCTGTATGTGCTCCATTGATGCAGC-3′ (seq. #2) wherein the sequence isalso effective. Compositions comprising cationic liposomes containing atleast one non-toxic cationic lipid, phosphatidylcholine and cholesterolmay be used as a carrier system.

DESCRIPTION OF THE INVENTION

[0007] The search for clinically useful radiation sensitizers fortreatment of cancers which fail to respond to radiation therapy has beenactively pursued. This invention provides specific sequences which,while inducing radiation sensitivity on tumor cells, is non-toxic tonormal tissue. As little as 10 ρmol/μl of the sequences encapsulated inliposomes is effective when tumor cells are contacted with thecompositions. It was found that the expression and enzymatic activity ofRaf-1 protein are inhibited in cells exposed to raf antisenseoligodeoxyribonucleotide (As-ODNs) directed against the translationinitiation site of human c-raf-1 cDNA. In contrast, treatment of cellswith an equimolar concentration of raf sense oligodeoxyribonucleotide(S-ODNs) had no effect on the expression and activity of Raf-1.Furthermore, it was observed radiosensitization of raf As-ODNs-treatedSQ-20B cells. The dose modifying factor of As-ODNs treatment was 1.4.This demonstrates that raf As-ODNs is a DNA sequence-specificradiosensitizer which may have potential for use in the radiationtherapy of cancers. Hence, the method of the invention comprisesadministration of a radiosensitizing effective amount of at least oneantisense nucleotide of no more than 40 bases containing the sequence5′-GTGCTCCATTGATGC-3′.

[0008] This invention provides new liposomal compositions which providemeans of enhancing the effect of oligonucleotides encapsulated in thenovel liposomes. The invention is exemplified using encapsulated rafoligodeoxyribonucleotides. The novel cationic liposomes of the inventionwere prepared using dimethyldioctadecyl ammonium bromide,phosphatidylcholine and cholesterol. However, other nontoxic cationiclipids such as N-(2,3-(dioleoyloxy)propyl)-N,N,N-trimethyl ammoniumchloride or1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadecenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride may be used. These liposomes provide protection fromdegradation in plasma and normal tissues to protect the oligonucleotideswhile they are reaching their intended target cells. Hence, smalleramounts of oligonucleotides are needed to obtain desired results.

[0009] Cationic liposomes have been used to deliver genes in vitro andin vivo (Felgner, Editorial, Human Gene Therapy 7:1791-1793, 1996). Thenovel formulation of the cationic liposomes to encapsudlate antisenseraf oligonucleotides has been tested and found effective. It has beenfound that these liposomes encapsulate >90% oligos. Liposomalencapsulation provides protection of antisense raf oligonucleotide fromdegradation in plasma, and normal tissues, and that tumor cells treatedwith the liposome-encapsulated antisense raf oligo (LE-ATG-AS raf ODN)are significantly radiosensitive compared to control or sense rafoligo-treated cells. It is now disclosed herein that LE-ATG-AS raf ODNinhibits Raf-1 protein expression in solid tumors. (Gokhale et al.,“Antisense 97: Targeting the Molecular Basis of Disease,” CambridgeSymposium Meeting, May 1997). The liposomal compositions of theinvention disclosed herein are believed to be particularly useful asradiosensitizers in solid tumors.

[0010] Materials and methods

[0011] oligodeoxyribonucleotides

[0012] The sense and antisense raf ODNs were designed against thetranslation initiation site of human c-raf-1 CDNA in accord with theteachings of Bonner (Bonner et al., Nucleic Acids Res., 14:1009-1015,1986), and have the following sequence: sense ODN (ATG-S raf),5′-GCATCAATGGAGCAC-3′ (seq. #3); antisense ODN (ATG-AS raf),5′-GTGCTCCATTGATGC-3′ (seq. #1), only two of the bases, one at each end,are phosphorothioated. While antisense sequences of raf of up to 40bases containing seq. #1 may be used, the larger sequences may be lesseffective. The fully phosphorothioated sequences may also be effective,but are more likely to cause toxic effects. That the sequences havingonly the end bases phosphorothioated are non-toxic to normal cellsgreatly enhances the value of such sequences for use in targetingmalignant cells.

[0013] Synthesis and purification of ODNs

[0014] Oligodeoxyribonucleotide synthesis was performed at LofstrandLabs Limited, Gaithersburg, using Beta-Cyanoethyl Phosphoramiditechemistry on Biosearch 8750 DNA synthesizers. Desired base linkages weremodified to phosphorothioate groups using3H-1,2-benzodithiole-3-1,1,1-dioxide as the sulfurizing agent. oligossynthesized at the 15μmol scale were cleaved and deprotected in 30%ammonium hydroxide for 24 hours at room temperature and purified overreverse phase chromatography columns. Deprotected DMT-On (trytl on)oligos retained by the support column as failure sequences were washedoff in basic aqueous solution. Full length product was detritylatedusing 2% trifluoro-acetic acid, washed with sterile, deionized water andeluted with 20% acetonitrile, dried and resuspended in sterile,deionized water. For quality control, a small aliquot of each oligopreparation was ³²P-end labeled and visualized by polyacrylamide gelelectrophoresis (20% acrylamide and 5% bis) followed by densitometerscanning of the labeled products.

[0015] Source of Cells

[0016] The SQ-20B tumor cells used were established in culture from thelaryngeal squamous carcinoma of a patient who had failed a full courseof radiation therapy. In vitro radiation survival analysis has confirmedthat these tumor cells are relatively radioresistant. Previously, it hadbeen demonstrated that the transfection of antisense human c-raf-1 cDNAinto SQ-20B cells leads to the down-regulation of endogenous raf-1 geneexpression, delayed tumor growth in athymic mice, and enhanced radiationsensitivity compared with the sense c-raf-1 cDNA transfectants, and theuntransfected tumor cells. Hence a “dual” role was proposed for Raf-1 inthese tumor cells; a direct role in the expression of the malignantphenotype, and an indirect role in cellular responses to radiationdamage.

[0017] Cell culture, cell viability and cell cycle assay

[0018] SQ-20B stock cultures were grown and maintained in completeDulbecco's modified MEM (GIBCO/BRL) containing 20% heat inactivatedfetal bovine serum (FBS), 2 mM glutamine, 0.1 mM non-essential aminoacids, 0.4 μg/ml hydrocortisone, 100 μg/ml streptomycin and, 100 U/mlpenicillin. For cell viability and cell cycle analysis, logarithmicallygrowing cells were cultured in T-25 flasks, complete medium was replacedwith 1% FBS containing medium in the presence of a desired concentrationof ODNs followed by continued incubation for various times. Controlcells were grown in 1% FBS containing medium without ODNs. Cells werecollected by trypsinization and viability was determined by the trypanblue dye exclusion assay. Duplicate samples were analyzed by the FACSmethod to determine the % distribution of cells in different phases ofthe cell cycle.

[0019] Intracellular uptake and stability of ODNs

[0020] Stock solutions of oligos were prepared by reconstitution of thelyophilized compounds in sterile phosphate buffered saline (PBS) justbefore use. Oligos (10 pmol/μl) were 5′-end labeled with [y-³²P]ATP (50μCi, 3000 Curies/mmol) and T4 polynucleotide kinase (10 U/μl), andpurified on ChromaSpin-10 column (Clontech). Logarithmically growingcells were rinsed with Hank's balanced salt solution and fresh mediumcontaining 1% FBS, 100 pmol/μl ODNS, and radiolabeled ODNs (2×10⁶ cpm)was added. Following incubation at 37° C. in a humidified, 5% Co₂atmosphere for the desired time, cells were collected, washed threetimes in PBS, and lysed for 2 hours at 37° C. in the buffer containing10 mM Tris-HCl (pH 7.5), 1% SDS, and 200 μg/ml proteinase K. DNAisolation was performed using the phenol:chloroform:isoamyl-alchohol(25:24:1) extraction procedure. ODNs uptake was determined by liquidscintillation counting of the aqueous phase, and was expressed as apercentage of the total radioactivity applied to the cells. Aliquots ofthe cell extracts (5×10⁷ cpm per sample) were resuspended in samplebuffer (95% formamide, 0.05% xylene cyanol, 0.05% bromophenol blue) andelectrophoresed on 8 M Urea-20% polyacrylamide gel. The dried gel wasauto-radiographed to visualize the oligos.

[0021] Raf-1 immunoprecipitation, immunoblotting, and in vitro kinaseactivity assays

[0022] Cells were washed twice with cold PBS and lysed at 4° C. for 10min. in RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5% sodiumdeoxycholate, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 20μg/mlaprotinin, 20μg/ml leupeptin). Insoluble material was removed bycentrifugation at 4° C. for 30 min at 9,000 xg and protein concentrationwas determined (Pierce). Immunoprecipitation was performed by incubatingthe lysate with polyclonal Raf-1 antibody directed against the last 12amino acids of human Raf-1 and conjugated with Protein A-Agarose (SantaCruz). Immunoprecipitates were washed once with the lysis buffer, twicewith 0.5M LiCl-0.1M Tris (pH7.4), and once with 10 mM Tris (pH 7.4). Forimmunoblotting, the immune-complex was boiled in Lammeli sample bufferand electrophoresed on a 7.5% SDS-poly-acrylamide gel, followed bywestern blotting using Raf-1 antibody and detection of Raf-1 protein bythe ECL method according to the manufacturer's protocol (Amersham).Raf-1 protein kinase activity was determined in vitro using apseudosubstrate peptide (Syntide 2, Santa Cruz) as substrate. Thephosphotransferase assay was performed by incubating Raf-1immunoprecipitates along with the exogenous substrate for 20 min at 30°C. in 40 μl of reaction buffer containing 25 mM HEPES (pH7.4), 25 mM 8glycerol phosphate, 1 mM DTT, 10 mM MnCl₂, 100 μM ATP, and 10 μCi of[Y-^(32P]ATP. The assay was terminated by spotting) 20 μl of thereaction mix onto 2 cm × 3 cm pieces of Whatman P81 phosphocellulosepaper. The filters were washed four times for 15 min in a solution of0.85% phosphoric acid. The Syntide 2-associated ³²p radioactivity boundto the filters was quantitated by Cerenkov counting.

[0023] Clonogenic radiation survival assay, and data analysis

[0024] The appropriate number of tumor cells were seeded into T-25flasks in complete medium containing 20% FBS. Cells were allowed toattach for 8 hr and the medium was replaced with medium containing 1%FBS and 100 pmol/μl of raf S- or As-ODNs. Control cells were incubatedwith complete medium containing 1% FBS. Oligo treatment lasted for 10hr, followed by exposure of the cells to the indicated graded doses ofγ-radiation. Irradiations were performed using a ¹³⁷Cs gamma irradiator(JL Shepard MARK I irradiator) at a dose rate of 3.83 Gy/min. Theirradiated cells were then maintained under these incubation conditionsfor an additional 2 hr. The growth medium in all flasks then wasreplaced with complete medium containing 20% FBS and the cells wereincubated for 7-10 days. Surviving colonies were fixed and stained with1% methylene blue. Colonies greater than 50 cells were scored and thedata were fitted to the Albright's computer-generated single-hitmultitarget and linear-quadratic models of radiation survival response.

[0025] Liposome preparation

[0026] Liposome-encapsulated raf oligodeoxyribonucleotides, LE-ATG-S rafODN and LE-ATG-AS raf ODN, were prepared using dimethyldioctadecylammonium bromide, phosphatidylcholine and cholesterol (Avanti PolarLipids, Inc., Alabaster, AL, USA) in a molar ratio of 1:3.2:1.6.Briefly, the lipids dissolved in chloroform or methanol were evaporatedto dryness in a round-bottomed flask using a rotatory vacuum evaporator.The dried lipid film was hydrated overnight at 4° C. by adding 1 ml ofODN at 1.0 mg/ml in phosphate buffered saline (PBS). The film wasdispersed by vigorous vortexing and the liposome suspension wassonicated for 5 min in a bath type sonicator (Laboratory Supplies Co.Inc., Hicksville, NY, USA). The ODN to lipid ratio was 30 μg ODN/mg oflipid. The unencapsulated ODN was removed by washing the liposomes bycentrifugation (3 times at 75,000 g for 30 min) in PBS. Theencapsulation efficiency was determined by the scintillation counting ofan aliquot of the preparation in which traces of ³²P-end labeled ODNwere added to the initial ODN. The entrapment efficiency was found tobe >90% (n=10). The liposome encapsulated ODN were stored at 4° C. andused within 2 weeks of preparation. Blank liposomes were preparedexactly as described above but without ODN.

[0027] Animals

[0028] Male Balb/c nu/nu mice, 10-12 weeks old, were maintained in theRRF facility of the Georgetown University according to accreditedprocedure and fed purina chow and water ad libitum.

[0029] Pharmacological disposition studies

[0030] The pharmacological disposition of free (ATG-AS) orliposome-encapsulated antisense raf oligodeoxyribonucleotide (LE-ATG-AS)was carried out in Balb/c nu/nu mice. Male Balb/c nu/nu mice wereinjected intravenously via tail vein with 30 mg/kg of ATG-AS raf ODN orLE-ATG-AS raf ODN. At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 hand 48 h after injection, one animal in each group was bled from theretro-orbital sinus into heparinized tubes and sacrificed by cervicaldislocation. The blood was centrifuged immediately at 2000 r.p.m for 10min at 4° C. to separate the plasma. The liver, spleen, kidney, heartand lung were rapidly excised and rinsed in ice-cold normal saline. Theorgans and plasma were stored frozen at −70° C. until analysis.

[0031] ODN was isolated from plasma samples using the phenol/chloroformextraction method, and from tissues using a DNA extraction kit(Stratagene, La Jolla, CA, USA). The extracts were then loaded onto 20%polyacrylamide/8 M urea gels and electrophoresed in TBE buffer. The gelwas electroblotted onto nylon membrane in 0.5X TBE buffer at 20 V for 1h. The blots were probed with ³²p-labeled sense raf ODN (ATG-S raf ODN)in Quickhyb buffer (Stratagene, La Jolla, Calif., USA) at 30° C.overnight. The ODN concentration standard was prepared by spiking knownamount of the ATG-AS raf ODN in blank plasma or blank tissue samples,followed by extraction as described above. The autoradiographs werescanned using a computer program (ImageQuant software version 3.3,Molecular Dynamics), and the amounts of ATG-AS raf ODN in varioussamples were calculated by comparison to standards.

[0032] SQ-20B tumor xenograft studies

[0033] Logarithmically growing SQ-20B cells (2×10⁶) were injectedsubcutaneously in the flank region on both sides in male Balb/c nu/numice under mild anesthesia. Tumors were allowed to grow to a mean tumorvolume of 115 mm³ before initiation of ODN treatment.

[0034] For intratumoral delivery of LE-ATG-AS raf ODN or LE-ATG-S rafODN, mice were randomly divided into 3 groups. Three mice in each groupreceived intratumoral injections of 4 mg/kg LE-ATG-AS raf ODN on theright flank, and LE-ATG-S raf ODN on the left flank. The ODN wasadministered intratumorally daily for 7 days. Control groups receivednormal saline or blank liposomes. Mice were sacrificed 24 hours afterthe last dose of ODN, and the organs were rapidly excised, rinsed inice-cold normal saline and stored at −70° C. until analysis. Raf-1protein expression was analyzed from tissue homogenates byimmunoprecipitation and immunoblotting and quantified using theImageQuant computer program as described above.

[0035] Results

[0036] Effects of raf ODNs on cell viability and cell cycledistribution, and intracellular uptake

[0037] ODNs sequences were directed towards the translation initiationsite of human c-raf-1 cDNA (15-mer, ends-phosphorothioated, S/As;25-mer, fully-phosphorothioated, S-F/As-F). Since cell survival inresponse to y-radiation is regulated during the cell cycle, and sinceseveral reports have suggested the possibility of a cell cycle controlof Raf-1 protein kinase activity, it was important to first determinethe effects of As-ODNs on the cell cycle distribution pattern of SQ-20Bcells. Logarithmically growing cells were exposed to 25-100 pmol/μl ofvarious ODNs for 4-12 hr, and the cell viability and cell cycledistribution patterns were measured. Sense and antisense ODNs showedtoxicity as compared to the control cells in 1% FBS-medium withoutoligos. The cell cycle distributions of ODNs-treated cells were alsofound to differ as compared to control cells. These data are in generalagreement with previous reports suggesting non-specific effects due tothe introduction of oligos per se. Remarkably, ends-modified As-ODNs wasneither cytotoxic nor had any significant effect on the cell cycledistribution profile of SQ-20B cells as compared to sense ODNs (S-ODNs).Similar observations were made with As-F and S-F ODNs. The intracellularuptakes of ODNs were examined. A linear increase in the intracellularlevel of ODNs was seen at between 2-12 hr post-treatment. Approximately4% (equivalent to 4 pmol/μl) of the total extracellular or applied ODNswas taken up by these tumor cells by 12 hr. Intracellular ODNs were alsofound to be stable as 15-mers.

[0038] Specificity of inhibition of Raf-1.

[0039] To ascertain the specificity of inhibition of Raf-1 proteinkinase by raf As-ODNs, all experiments were performed, in parallel,using two controls: i) cells were exposed to raf S-ODNs using treatmentconditions identical to As-ODNs to rule out the influence ofnon-specific effects due to ODNs; and ii) control cells were treatedwith 1% FBS containing medium (without ODNs) to determine the base-linelevels of the Raf-1 expression and enzymatic activity in SQ-20B cells.

[0040] The dose-dependence and time-course of inhibition of Raf-10protein expression by As-ODNs was studied using a combination of Raf-1immunoprecipitation and immunoblotting assays. Densitometric analysis ofthe Raf-1 band (˜75 kDa) showed that about 40% inhibition of Raf-1protein expression occurred at 12 hr when 100 pmol/μl As-ODNs wasapplied to the cells. Raf-1 protein expression in the S-ODNs-treatedcells (extracellular 100 pmol/μl, 12 hr) was found to be identical tothe expression in untreated control cells, and in cells exposed to 25pmol/μl extracellular As-ODNs. Specificity of the Raf-1 antibody wasconfirmed by elimination of the ˜75 kDa band observed in control cellswhen immunoprecipitation was performed with antigen-blocked antibody.Time-course experiments revealed that approximately 50% inhibition ofRaf-1 protein was achieved by 12 hr post-incubation with 100 pmol/μlextracellular As-ODNs. The inhibitory effect of As-ODNs appeared todiminish by 18 hr. This recovery of the Raf-1 expression may beattributed to the apparent degradation of As-ODNs over time and to thesynthesis of new protein. Nevertheless, three independent studiesindicated that approximately 50% inhibition of Raf-1 protein occurred by12 20 hr in the As-ODNs-treated cells as compared to cells treated withequimolar concentration of S-ODNs.

[0041] Having established 100 pmol/μl as a non-toxic and inhibitoryextracellular dose of raf As-ODNs in SQ-20B cells relative to theequimolar concentration of S-ODNs, the effect of As-ODNs on in vitrophosphotransferase activity of Raf-1 protein kinase was examined.Consistent with immunoblotting data, approximately 50% inhibition of thein vitro Raf-1 protein kinase activity was noted in Raf-1immunoprecipitates of As-ODNs-treated cells as compared to theS-ODNs-treated cells (applied ODNS: 100 pmol/μl, 12 hours) and theuntreated control cells. Experiments were also performed to measure theRaf-1 immune-complex-associated in vitro kinase activity in tumor cellsexposed to the fully modified ODNs (S-F/As-F, applied dose 100 pmol/μl,12 hr). Inhibition of Raf-1 activity was observed in the As-FODNs-treated cells as compared to the S-F ODNs response. It isnoteworthy that the expression and activity of Raf-1 were observed to besimilar in the control (C, without oligo) and raf S-ODNs-treated cells(S). This finding along with the concurrent inhibition of the expressionand activity of Raf-1 noted in the raf As-ODNs-treated cells (As),implied that the inhibition of Raf-1 protein kinase wassequence-specific, and not due to the non-specific effects of ODNs.

[0042] raff As-ODNs is a biologic radiosensitizer of SQ-20B cellsRadiation survival dose responses of SQ-20B cells exposed to S-andAs-ODNs (100 pmol/μl, 12 hr) were evaluated. (S- and As-ODNs used inthis study do not contain the G-quartet or CpG motifs previously shownto be responsible for non-antisense-specific effects such as enhancedaffinity for protein or interference with the immune response.) Theplating efficiencies indicated that the As-ODNs treatment had no effecton cell viability as compared to S-ODNs-treated cells (Table 1). Thesedata are also in agreement with the S- and As-ODNs effects on theviability of logarithmically growing cells discussed earlier. Radiationsurvival dose responses of the control (without oligo) andS-ODNs-treated cells were almost identical. Most important, As-ODNstreatment resulted in decreases of the shoulder and the slope of thesurvival curve. The radiobiological parameters were obtained by fittingthe data (surviving number of colonies) to the single-hit multitarget(D, D_(q), n) and linear-quadratic (α, β) models of radiation survivalresponse. In addition, the value of a model-free parameter, meaninactivation dose (D) was calculated (14) (Table 1). Based on a ratio ofthe mean inactivation dose, the dose modifying factor (DMF) of As-ODNstreatment was ˜1.4. Significant decreases observed in the values ofradiobiological parameters, D, D_(q), and D₀ of SQ-20B cells followingtreatment with the raf As-ODNs indicate a good correlation between theDNA sequence-specific inhibition of Raf-1 protein kinase and theradiosensitization of these relatively radioresistant tumor cells.

[0043] The studies indicated that greater than 50% inhibition of Raf-1expression could be achieved with only 10 pmol/μl of theliposome-encapsulated raf As-ODNs.

[0044] Liposomal encapsulation protects ATG-AS raf ODN in wivo

[0045] The plasma concentration-time profile of LE-ATG-AS raf ODN wasstudied. Dosage of 30 mg/kg LE-ATG-AS raf ODN or ATG-AS raf ODN wasadministered i.v. in Balb/c nu/nu mice. Blood samples were collectedfrom retro-orbital sinus at indicated times after injection and the ODNin plasma samples was extracted by phenol:chloroform. The samples wereelectrophoresed on 20% polyacrylamide/8 M urea gel and electroblotted onnylon membrane. The blots were probed with ³²P-labeled ATG-S raf ODN.Auto-radiographs were scanned using a computer program (ImageQuantsoftware version 3.3, Molecular Dynamics). St, standard prepared byspiking known concentration of ATG-AS raf ODN in blank plasma.Quantification data were calculated based on a known concentration ofthe standard sample, and then normalized against dilution factors atvarious time points.

[0046] Following intravenous administration, the peak plasmaconcentration of 6.39 μg/ml was achieved and intact ODN could bedetected up to 24 h. The decrease in plasma concentration of LE-ATG-ASraf ODN followed a biexponential pattern with an initial half-life(t_(1/2α)) of 24.5 min and a terminal half-life (t_(1/2β) of) 11.36 h.The area under the plasma concentration-time curve for LE-ATG-AS raf ODNwas 5.99 μg.h/ml, with total body clearance of 75.94 ml/min/kg andvolume of distribution of 74.67 L/kg. In contrast, intact free ODN(ATG-AS raf ODN) was detectable only at 5 min; the plasma concentrationbeing 9.75 μg/ml. These observations indicate that ATG-AS ODN was eitherrapidly cleared from the circulation, or extensively degraded in plasmadue to nuclease activity.

[0047] Normal tissue distribution profiles of LE-ATG-AS raf ODN werestudied. Tissue samples were collected at indicated times after i.v.administration of 30 mg/kg LE-ATG-AS raf ODN. ODN was extracted fromhomogenized tissues using a DNA extraction kit (Stratagene). Sampleswere electrophoresed and electroblotted. The blots were probed with³²P-labeled ATG-S raf ODN and autoradiographs were analyzed.Quantification data were calculated based on a known concentration ofthe standard sample, and then normalized against the weight of thetissue sample collected.

[0048] Intact ODN could be detected in all organs examined up to 48hours after administration. Interestingly, following ATG-AS raf ODNadministration, intact ODN could be detected only at 5 minutespost-administration in all the organs examined and degradation productswere observed at all other times. These findings, along with the plasmadata for LE-ATG-AS raf ODN, suggests that ODN with only the end basesphosphorothioated is rapidly degraded in vivo and that liposomeencapsulation using the liposomes of the invention protects it fromdegradation for at least 48 hours. TABLE 1 Radiation survival parametersof SQ-20B cells treated with raf oligodeoxyribonucleotides raf No. of D₀Dq α β D ODNs Expts. (Gy) (Gy) n (Gy⁻¹) (Gy⁻²) (Gy) Sense/Control* 63.028 1.265 1.519 0.2373 0.0050 3.694 Antisense 3 2.374 0.551 1.2610.3614 0.0038 2.628

[0049] The appropriate number of cells were seeded into two replica T-25flasks per dose in each experiment. Control cells were cultured inmedium containing 1% FBS without oligo. Plating efficiencies of theS-ODNs-treated, As-ODNs-treated and control cells were in the range of13-58%, 28-61% and 41-67%, respectively. Clonogenic survival data werecomputer-fitted to the single-hit multitarget and the linear-quadraticmodels of radiation survival dose response. Composite values of thevarious parameters were obtained from the three experiments performedwith the S-ODNs-treated and the three experiments using the controlcells.

[0050] Specificity of inhibition of Raf-1 protein expression andactivity in vitro

[0051] Initially, the possibility of cytotoxic effects of liposomes inSQ-20B cells was examined. Blank liposomes, at concentration equivalentto 10 μM LE-ATG-AS raf ODN, were found to be non-cytotoxic as determinedby the clonogenic and trypan blue dye exclusion methods. However, blankliposomes showed cytotoxicity at doses higher than 20 μM. Therefore, adose of 10 μM or less was used for in vitro experiments. Further, 10 μMLE-ATG-AS raf ODN or LE-ATG-S raf ODN was non-toxic to SQ-20B cells.

[0052] Specificity of inhibition of Raf-1 protein expression byLE-ATG-AS raf ODN. Logarithmically growing SQ-20B cells were treatedwith 10 μM LE-ATG-AS raf ODN (AS), 10 μM LE-ATG-S raf ODN (S) or blankliposomes (BL) for indicated time in 1% FBS containing medium. Untreatedcontrol cells (C) were simultaneously switched to 1% FBS containingmedium for 8 hours. Whole cell lyates were normalized for total proteincontent and immunoprecipitated with agarose-conjugated polyclonalanti-Raf-1 antibody (Santa Cruz). Immune-complexes were resolved on 7.5%SDS-PAGE and Raf-1 protein expression was detected by immuno-blottingwith polyclonal anti-Raf-1 antibody (Santa Cruz), followed by the ECLdetection protocol (Amersham). Results from three independentexperiments were quantified using a computer program (ImageQuant,Molecular Dynamics), and data are expressed relative to the level ofRaf-1 in LE-ATG-S raf ODN-treated cells (bottom). Panel B: Dose-responseanalysis. Logarithmically growing SQ-20B tumor cells were treated withindicated concentrations of LE-ATG-AS raf ODN (AS) or LE-ATG-S raf ODN(S) in 1% FBS containing medium for 8 h. Normalized cell lysates wereanalyzed for Raf-1 protein expression.

[0053] Logarithmically growing SQ-20B cells were treated with 10 μMLE-ATG-AS raf ODN (AS), or 10 μM LE-ATG-S raf ODN (S) for 8 h in 1% FBScontaining medium. Control cells (C) were simultaneously switched to 1%FBS containing medium for 8 hours. Whole cell lysates were normalizedfor protein content, and Raf-1 was immunoprecipitated.Phosphotransferase activity of Raf-1 in immune-complexes was assayed invitro using a physiologic substrate MKK1. Radiolabeled reaction productswere separated by electrophoresis and autoradiographed.

[0054] Time-course experiments revealed that a maximum inhibition(52.3±5.7%) of Raf-1 protein expression (˜74 kDa) occurred at 8 hourspost-incubation of cells with 10 μM LE-ATG-AS raf ODN. The inhibitoryeffect of LE-ATG-AS raf ODN was maintained up to 24 h (45.6±9.8%). Thelevel of Raf-1 protein was comparable in the control untreated cells(C), blank liposome-treated. cells (BL), and LE-ATG-S ODN-treated cells,indicating the LE-ATG-AS raf ODN specifically inhibited the Raf-1protein expression in SQ-20B cells. Dose response studies showed that35.94±16.8% and 52.3±5.7% inhibition of Raf-1 expression occurred with 5μM and 10 μM LE-ATG-AS raf ODN treatment for 8 hours.

[0055] The effects of LE-ATG-AS raf ODN on the enzymatic activity ofRaf-1 protein kinase using mitogen-activated protein kinase kinase(MKKl) as substrate were studied. In concurrence with Raf-1 proteininhibition data, it was found that 10 μM LE-ATG-AS raf ODN treatment for8 hours inhibited 62.6±9.0% in vitro phosphotransferase activity ofRaf-1 protein. LE-ATG-S raf ODN did not have any effect on the Raf-1protein kinase activity as compared with the untreated control cells.

[0056] LE-ATG-AS raf ODN is a biological radiosensitizer

[0057] Radiation survival dose responses of SQ-20B cells exposed toLE-ATG-AS raf ODN, LE-ATG-S raf ODN, and blank liposomes are presentedin Table 2. Comparison of the radiation survival dose response of SQ-20Bcells treated with LE-ATG-AS raf ODN (AS), LE-ATG-S raf ODN (S), orblank liposomes (BL). The appropriate number of cells were seeded induplicate to obtain 40-60 colonies per T-25 flask (Costar) for eachradiation dose. The clonogenic survival data were computer-fitted to thesingle-hit multitarget model of radiation survival dose response.Representative data from one experiment performed for each treatmentcategory were evaluated.

[0058] The plating efficiencies of cells treated with S/AS ODN or blankliposomes were comparable (Table 2). Radiation survival dose responsesof the blank liposome-treated (BL) and LE-ATG-S raf ODN-treated cellswere also comparable. LE-ATG-AS raf ODN treatment resulted in asignificant radiosensitization (Table 2). Based on a ratio of the meaninactivation dose, the dose modifying factor (DMF) of LE-ATG-AS raf ODNtreatment was ˜1.6. Significant decreases observed in the values ofradiobiological parameters, {overscore (D)}, D_(q), and D₀ of SQ-20Bcells following treatment with the LE-ATG-AS raf ODN indicate a goodcorrelation between the DNA sequence-specific inhibition of Raf-1protein kinase and the radiosensitization of these relativelyradioresistant tumor cells.

[0059] LE-ATG-AS raf ODN is a specific inhibitor of Raf-1 proteinexpression in solid tumor

[0060] The effects of intratumoral administration of LE-ATG-AS raf ODNand LE-ATG-S raf ODN on the expression of Raf-l protein was examined ina SQ-20B tumor xenograft model. Mice with tumors on both flanks receivedintratumorally LE-ATG-AS raf ODN on the right flank, and LE-ATG-S ODN onthe left flank. Results demonstrated a significant inhibition of Raf-1protein in tumor tissue following treatment with LE-ATG-AS raf ODNcompared with LE-ATG-S raf ODN (60.3±6.4%).

[0061] Inhibition of Raf-1 protein expression by LE-ATG-AS raf ODN inSQ-20B tumor xenografts

[0062] SQ-20B tumors were established subcutaneously in both hind limbsof nude mice, Balb C nu/nu (mean tumor volume 115 mm³) Each animal thenreceived intratumoral injections of LE-ATG-AS raf ODN (AS) on the rightflank and LE-ATG-S raf ODN (S) on the left flank at a dose of 4 mg/kgdaily for 7 days. Tumor tissue was excised 24 h after the lasttreatment, and Raf-1 protein expression in tumor samples was analyzed.ECL images were quantified using a computer program (ImageQuant,Molecular Dynamics), and quantification data from three mice areexpressed as the level of Raf-1 protein expression in LE-ATG-AS raf ODN-treated tumors relative to LE-ATG-S raf ODN-treated tumors.

[0063] Liposomes prepared in accord with the teachings of the inventionare non-toxic both in culture and in animals. While only specificliposomes are disclosed herein, the novel carriers may be used fordelivery of a variety of DNA-based compounds for delivery. TABLE 2Radiation Survival Parameters of SQ-20B Cells Treated with LE-ATG-S/Asraf ODN raf ODN No. of Expts. D_(o)(Gy) D_(q)(Gy) {overscore (n)}α(Gy⁻¹) β(Gy⁻²) {overscore (D)}(Gy) Blank liposomes/ 5 2.795 1.445 2.0120.2184 0.0087 3.659 LE-ATG-S* ±0.38 ±1.22 ±1.34 ±0.11 ±0.00 ±0.02LE-ATG-AS 3 2.287 0.051 1.021 0.4385 0.0000 2.280 ±0.23 ±0.05 ±0.19±0.05 ±0.00 ±0.00

[0064] The appropriate number of cells were seeded in duplicate T25flasks per dose in each experiment. Plating efficiencies of the blankliposome-treated, LE-ATG-S raf ODN-treated, and LE-ATG-AS rafODN-treated cells were in the range of 65-79%, 52-90% respectively.Clonogenic survival data were computer-fitted to the single-hitmultitarget and the linear-quadratic models of radiation survival doseresponse.

[0065] The liposomes of the invention provide significant protection ofantisense oligonucleotides against degradation in blood and normaltissue. The formulation may replace the need for complete modificationof all bases of the antisense oligonucleotides for therapeutic uses.Compositions comprising oligonucleotides may be administered in manyways, depending on the target tissue.

[0066] The particular method used to deliver compositions of theinvention to the tissues of the intact animal will depend on theparticular tissue to which it is administered. For example, compositionsof the invention can be administered intrathecally to facilitate contactof the active agent with neuronal tissue. For administration to thelung, the liposomal compositions may be administered transbronchially asa spray or mist. Liposomal compositions may also be administered totissue locally during surgery. For example, the compositions could beadministered into the peritoneal cavity as a mist during surgery.

[0067] The liposomes may also be injected into the target tissue or intothe arterial blood supply to the target tissue. When the target tissueis the lining of a hollow organ, they may be introduced into the lumenof the organ.

[0068] The dosage required will depend on the agent and the subjectbeing treated with the liposomal compositions. For example, when aradiosensitizing oligonucleotide is administered by means of liposomes,a radiosensitizing amount of oligonucleotides must reach the targetorgan.

[0069] The radiosensitizing oligonucleotides may also be administeredsystemically. A preferred method of administration is by intravenousinjection.

[0070] Acceptable carriers include, for example, glucose, saline,phosphate buffered saline. Carriers may also contain other substancesfrequently found in pharmaceuticals such as preservatives, emulsifiersand surfactants.

[0071] Dosage will depend on the extent to which it is possible topresent the active agents to the target tissue. The appropriate dosageshould deliver a serum concentration of about 1 μg/ml to 1000 μg/ml. Insome instances, this dosage can be delivered into the target tissuedirectly. Hence, that level of dosage need not be achieved in the totalblood volume.

1 3 15 base pairs nucleic acid single unknown DNA (genomic) NO YES 1GTGCTCCATT GATGC 15 25 base pairs nucleic acid single unknown DNA(genomic) NO YES 2 CCTGTATGTG CTCCATTGAT GCAGC 25 15 base pairs nucleicacid single unknown DNA (genomic) NO YES 3 GCATCAATGG AGCAC 15

What we claim is:
 1. A composition comprising a cationic liposomecontaining a cationic lipid, phosphatidylcholine and cholestrol.
 2. Acomposition of claim 1 wherein the liposome contains an antisenseoligonucleotide sequence.
 3. A composition of claim 2 wherein theantisense sequence is a raf oligodeoxynucleotide.
 4. A composition ofclaim 3 wherein the antisense sequence is of the formula5′-GTGCTCCCATTGATGC-3′ wherein only the terminal sequences arephosphorothioated.
 5. A composition of claim 1 in a pharmaceuticallyacceptable carrier.
 6. A composition of claim 4 in a pharmaceuticallyacceptable carrier.
 7. A composition of claim 1 wherein thepharmaceutically acceptable carrier is isotonic.
 8. A composition ofclaim 4 wherein the pharmaceutically acceptable carrier is a buffered,isotonic solution.
 9. A method of radiosensitizing tumor tissue byadministration of a radiosensitizing effective amount of at least oneantisense oligonucleotide of no more than 40 bases containing thesequence 5′-GTGCTCCATTGATGC-3′.
 10. A method of claim 9 wherein theoligonucleotide is phosphorothioated at only the end nucleotides.
 11. Amethod of claim 9 wherein the oligonucleotide is phosphrothioated atonly the end nucleotides.
 12. A method of claim 9 wherein theoligonucleotide is administered intravenously.
 13. A method of claim 9wherein the oligonucleotide is administered directly to the targettissue.
 14. A method of claim 9 wherein the oligonucleotide isadministered into the arterial supply to the target tissue.
 15. A methodof claim 9 wherein the oligonucleotide is of the formula5′-GTGCTCCATTGATGC-3′ and only the end bases only are phosphorothioted.16. A composition of matter comprising liposomes containing the sequence5′-GTGCTCCATTGATGC-3′ in a pharmaceutically acceptable carrier.
 17. Acomposition of claim 1 wherein the cationic lipid is dimethyldioctadecylammonium bromide.